Down-regulation Of COX? Promotes Metastasis Of Hepatocellular Carcinoma Via ROS/CXCR4 Signaling Pathway

Background:Liver cancer ranks sixth in cancer incidence and second in cancer mortality.In China,the number of people who died of liver cancer reaches 369,000every year,and the overall 5-year survival rate is only 12.1%.Metastasis is the main cause of liver cancer death.Exploring its mechanism has become a key problem in the prevention and treatment of liver cancer.Previous studies have found that mitochondrial dysfunction is closely related to tumor metastasis.Cytochrome C oxidase subunit4?cytochrome c oxidase subunit 4,COX??is an important component of mitochondrial respiratory chain,and its abnormal expression can lead to mitochondrial dysfunction.It is not clear whether it can promote the metastasis of liver cancer.By means of in vivo and in vitro experiments,we will explore the effect of COX?expression reduction on liver cancer metastasis and clarify its molecular mechanism.Objective:1.The expression of COX?in liver cancer tissues was determined by quantitative COX?in liver cancer and adjacent tissues.And the effect of COX?expression intensity on the prognosis of patients was clarified by analyzing the clinicopathological parameters of liver cancer patients.2.And we conducted in vivo and in vitro experiments to clarify the specific function of COX?expression in the invasion and migration of hepatocellular cells.3.Confirm the relationship between COX?and CXCR4 in the mechanism of invasion and metastasis of liver cancer through experimental data.4.Clarify the specific relationship among COX??mitochondrial ROS and CXCR4in the mechanism of invasion and metastasis of HCC through experimental data.Methods:1.QPCR,Western-blot and immunohistochemistry were used to detect the differences in the expression of COX?in liver cancer and adjacent tissues,and the correlation between the expression level of COX?in liver cancer tissues or the prognosis of liver cancer patients was statistically analyzed.2.After interfering with the expression of COX?in SNU-368 and SNU-739hepatocellular carcinoma cell lines,the effect of reducing the expression of COX?in promoting invasion and metastasis of hepatocellular carcinoma cells was verified in vitro by using scratch and Transwell^TM experiments.A COX?interference-stabilized hepatocellular carcinoma cell line with luciferase was constructed,and a tumor-bearing model was constructed in nude mice.3.After interference of COX?expression in hepatocellular carcinoma cell lines SNU-368 and SNU-739,the effect of decreased COX?expression on CXCR4expression was detected by Western-blot and immunofluorescence.After blocking the effect of CXCR4 with an inhibitor,the Transwell^TM assay examined whether changes in the invasion and migration capacity of HCC cells induced by COX?interference were affected.4.MitoSOX and flow cytometry were used to detect the changes of mitochondrial ROS levels in hepatocellular cells after COX?interference.After reducing the intracellular ROS levels with reducing agents,Western-blot was used to detect whether the changes in CXCR4 expression caused by COX?interference were affected.Results:1.The results of qPCR,Western-blot and immunohistochemistry showed that the expression of COX?in liver cancer tissues was observably decreased when compared with that in paracancer tissues.The results of statistical analysis showed that the expression of COX?in liver cancer tissues was markedly correlated with the PVTT,TNM stage and tissue differentiation of liver cancer.The disease-free survival and overall survival of HCC patients with low COX?expression were dramatically lower than those with high COX?expression.2.In vitro and in vivo experiments confirmed the interference with COX?expression significantly enhanced the invasion and metastasis of HCC cells.3.After interfering with the expression of COX?in HCC cells,the expression level of CXCR4 was significantly increased.Blocking the effect of CXCR4 with inhibitors could memorably inhibit the increased invasion and metastasis of HCC cells caused by COX?interference.4.After interfering with the expression of COX?in liver cancer cells,the level of mitochondrial reactive oxygen increased significantly;The use of reducing agent can markedly block the rising of CXCR4 expression caused by COX?interference after reducing intracellular ROS level.Conclusion:The expression of COX?in hepatocellular carcinoma tissues is significantly lower than which in paracancer tissues,and the decreased COX?expression enhance the invasive migration of hepatocellular cells by way of promoting mitochondrial ROS to up-regulation of CXCR4.As a result,the lower expression of COX?is always related to worse prognosis.This essay reveal a new mechanism of liver cancer metastasis and provide a new potential target for the clinical prevention and treatment of hepatocellular.